CN101705283A - Targeting medicament curative effect related gene mRNA expressive level detection Luminex and detection method - Google Patents

Abstract

The invention discloses a receptor tyrosine kinases inhibiting type targeting medicament curative effect related gene mRNA expressive level detection Luminex. The detection Luminex mainly comprises a micro-ball, supporting extending probes, and amplification extension probes. The micro-ball is coupled with a supporting probe modified by amino; the supporting extending probes are used for connecting the supporting probe and a target gene mRNA, and each supporting extending probe comprises a specific sequence the terminal 5' of which can combine with a corresponding target gene mRNA, a spacer arm sequence, and a sequence the terminal 3' of which can complementarily pair with the corresponding specific sequence of the supporting probe at; and the amplification extension probes are used for connecting the target gene mRNA and information detection components, and each amplification extension probe comprises a specific sequence the terminal 5' of which can combine with the corresponding target gene mRNA, a spacer arm sequence, and a sequence the terminal 3' of which can complementarily pair with a markup probe. The invention also discloses a detection method of the receptor tyrosine kinases inhibiting type targeting medicament curative effect related gene mRNA expressive level by using the liminex. In the invention, the process can be carried out without the steps of RNA extraction, reverse transcription, PCR and the like and the detection result is influenced less by the quality of RNA in the sample, thereby the accuracy of the detection result is guaranteed; on the other hand, because the liminex technology enables a parallel multi-index detection to be realized, multiple inside genes can be set in one detection, thereby the detection result is more reliable.

Description

The targeting medicament curative effect related gene mRNA expression level detects liquid-phase chip and detection method

Technical field

The invention belongs to biology field, relate to medical science and biotechnology, the concrete receptor tyrosine kinase inhibitors class targeting medicament curative effect related gene mRNA expression level that relates to detects liquid-phase chip and detection method.

Background technology

Tyrosylprotein kinase has occupied crucial status in the intracellular signal transduction path, regulating a series of physiological processs such as growth in the cell paste, differentiation, death, and it is disorderly that its unconventionality expression will cause cell proliferation to be regulated, and then causes tumour to take place.In addition, the unconventionality expression of Tyrosylprotein kinase is also closely related with the chemotherapy resistance of the invasion and attack of tumour and transfer, tumor neovasculature generation, tumour, is that target spot carries out medicament research and development and become the focus of antitumor drug research in the world with the Tyrosylprotein kinase.

Tyrosylprotein kinase comprises two kinds of receptor type and non-receptor types, and (receptor tyrosine kinase RTKs) is maximum class of enzymes connection acceptor to receptor tyrosine kinase, it is an acceptor, be again enzyme, can combine with part, and with the tyrosine residues phosphorylation of target protein.Found the RTKs that kind more than 50 is different, main type comprises: 1. Urogastron (epidermal growth factor, EGF) acceptor comprises members such as EGFR, HER2, ErbB4; 2. platelet-derived growth factor (platelet-derived growthfactor, PDGF) acceptor, comprise PDGFR α, PDGFR β, colony-stimulating factor 1 acceptor, stem cell factor acceptor (Stem cell factor receptor, SCFR/KIT), member such as FLK2/FLT3; 3. Regular Insulin and insulin-like growth factor-i (insulin and insulin-like growth factor-1, IGF-1) acceptor, comprise that (insulin-like growth factor I receptor IGF-1R) waits the member to the type-1 insulin like growth factor acceptor; 4. nerve growth factor (nerve growth factor, NGF) acceptor; 5. fibroblast growth factor (fibroblast growth factor, FGF) acceptor; 6. (vascularendothelial growth factor, VEGF) acceptor comprises vascular endothelial growth factor: VEGFR1 (FLT-1), VEGFR2 (KDR/Flk-1), VEGFR3 members such as (Flt-4).

At present, the target therapeutic agent of oncotherapy mainly contains two classes, and a class is receptor tyrosine kinase inhibitors class targeted drug (Tyrosine Kinase Inhibitor, TKI), mainly comprise Gefitinib (Gefitinib, Ireasa), Tarceva (Erlotinib, Taceva) etc.; Another kind of is the monoclonal antibodies targeted drug, comprises Cetuximab (cetuximab), handkerchief Buddhist nun monoclonal antibody (Vectibix) etc.Receptor tyrosine kinase inhibitors class targeted drug combines with acceptor born of the same parents inner segment kinases land by diffusing into tumour cell, thereby suppresses the phosphorylation of acceptor, the conduction of blocking-up acceptor downstream signal path, performance antitumor action.

Though the curative effect of TKI class targeted drug is approved that extensively the individual difference of drug effect is very big after patient's medication.Vast amount of clinical is verified, and curative effect/toxic side effect of TKI is directly related with the mRNA expression level of some receptor tyrosine kinase genes involved in patient's body, and they are respectively:

(1) targeted therapy and EGF-R ELISA (EGFR)

EGF-R ELISA (EGFR) is the main target of targeted therapy, the targeted drug that acts on EGFR that FDA has ratified comprises EGFR tyrosine kinase inhibitor (EGFR-TKI) and anti-egfr antibodies medicine. but that clinical application shows these targeted drugs is only effective to the part patient. and clinical study is verified: with the EGFR path is that the EGFR mrna expression is closely related in the targeting medicament curative effect of target and the tumor tissues, be that the high patient of EGFR gene expression dose is to the targeted drug sensitivity, it is lower that otherwise the patient that expression level is low shows drug effect. therefore, American National cancer integrated network (NCCN) was recommended in the clinical guidelines in 2009: the patient is before accepting the targeted drug treatment, carrying out the EGFR gene expression dose detects, determine whether to accept targeted therapy, reduce medical expense, save the valuable treatment time.

(2) imatinib and stem cell factor acceptor (Stem cell factor receptor, SCFR/KIT)

Stem cell factor acceptor (Stem cell factor receptor, SCFR/KIT) be one of platelet-derived growth factor acceptor (PDGFR) family member, the KIT albumen of c-KIT proto-oncogene coding is a kind of transmembrane glycoprotein, belongs to receptor tyrosine kinase family.Studies show that KIT gene expression product and part thereof are the important regulating and controlling factor that human multiple histocyte grows, and be closely related with the generation of tumour.Imatinib mesylate (Imatinib mesylate, STI571, imatinib mesylate), the derivative of genus 2-phenyl-aminopyrimidine is the oral receptor tyrosine kinase inhibitors of a kind of small molecules.Be used for clinically in 2002 through FDA approval, be successfully applied to gastrointestinal stromal tumor (GIST) and chronic granulocytic leukemia (CML), clinical efficacy is exciting.Its mechanism of action is that medicine is incorporated into the ATP-binding site of Tyrosylprotein kinase functional zone in the KIT albumen endochylema, blocks phosphate group by the transfer of ATP to the substrate tyrosine residues, thereby suppresses cell proliferation and recover the apoptosis program.

The KIT protein expression is all arranged in most of tumour cells, and expression level is widely different.Clinical study confirmed KIT expression level and gastrointestinal stromal tumor imatinib curative effect and has been proportionate lifetime, and promptly the patient that expression level is high is to the imatinib medicaments insensitive, otherwise the low patient of expression level shows the part resistance.Therefore, the ASCO report is special reminds preceding must the detection of gastrointestinal stromal tumor application imatinib to confirm the KIT positive.

(3) targeted therapy and type-1 insulin like growth factor acceptor (insulin-like growth factor I receptor, IGF-1R)

Type-1 insulin like growth factor acceptor (IGF-1R) is the important target of targeted therapy, and the signal transduction pathway of IGF-1R and the generation of tumour are closely related.Suppress the activity of IGF-1R, can effectively control the growth and the transfer of tumour cell, strengthen the susceptibility of tumour chemotherapy, radiotherapy.The treatment that with IGF-1R is target mainly contains IGF-1R monoclonal antibody, antisense oligonucleotide, IGF-1R monoclonal antibody, small molecules kinase inhibitor etc.The signal transduction of IGF-1R also can cause the malignization of cell and the change of cell adhesion.IGF-1R has become the focus target of specific treatment tumour at present.A large amount of clinical studyes show that IGF-IR presents high expression level in malignant tumor tissues such as colorectal carcinoma, prostate cancer, mammary cancer, nonsmall-cell lung cancer, the esophageal carcinoma, cancer of the stomach.Clinical studies show: IGF-1R and the NSCLC patient significant correlation alive that lives forever through Gefitinib treatment.Tumour patient detects IGF-1R mRNA expression level, to the selection of targeted drug clinically, dosage, targeted drug susceptibility determine and the judgement of prognosis has great significance, improve the validity of targeted therapy simultaneously, for the patient strives for treatment time of more preciousnesses significantly saving the treatment expense.

(4) targeted drug and vascular endothelial growth factor receptor (VEGFR)

Vasculogenesis is the prerequisite of human tumor growth and transfer, and the vasculogenesis that suppresses the tumour cell mediation has become the important research direction of seeking new type antineoplastic medicine in recent years.Vascular endothelial growth factor (VEGF) and acceptor (VEGFR) thereof are the main targets of angiogenesis inhibitor targeted therapy.The drug mechanism of angiogenesis inhibitor mainly is to reach the purpose that stops growth of tumor, transfer and recurrence by the vasculogenesis that suppresses the tumour cell mediation.This type of medicine of FDA approval at present comprises Sutent, Xarelto and rhuMAb-VEGF etc.

The member of VEGFR family mainly comprises: VEGFR1 (FLT-1), VEGFR2 (KDR/Flk-1), VEGFR3 (Flt-4), VEGFR1 plays a significant role in the hemopoietic process, and VEGFR2 plays main regulating effect in the raising of mitogenesis, vasculogenesis and VEGF perviousness effect.

Clinical study confirms, VEGFR2 can be used as colon cancer patient and uses independently predictor of 5-FU or capecitabine and oxaliplatin or rhuMAb-VEGF (FOLFOX/BV or XELOX/BV) combined treatment clinical efficacy, its treatment curative effect and VEGFR2 gene expression dose are closely related, be that the high patient's of VEGFR2 gene expression dose meta is longer lifetime that gets nowhere, lifetime is lower and the low patient's meta of VEGFR2 gene expression dose gets nowhere.Therefore the patient carried out the VEGFR gene expression dose and detects, thereby determine whether to accept targeted therapy before accepting the targeted drug treatment, can reduce medical expense, saved the valuable treatment time.

(5) targeted therapy and platelet-derived growth factor acceptor (PDGFRB)

Receptor tyrosine kinase inhibitors class medicine is to having the general name of the medicine that suppresses receptor tyrosine kinase activity, at present, the receptor tyrosine kinase inhibitors commonly used of target PDGFRB has Sutent and Suo La for Buddhist nun, imatinib, is present the most frequently used clinically tumor chemotherapeutic drug.Its pharmacological action mainly is: by suppressing the RAF/MEK/ERK signal transduction pathway, directly suppress tumor growth on the one hand; Block tumor neovasculature formation by suppressing PDGFR and VEGFR on the other hand, suppress the growth of tumour cell indirectly.

PDGFRB is the important member in the PDGFR signal transduction pathway, comprises two kinds of PDGFR α and PDGFR β, plays an important role at the aspects such as propagation, invasion and attack and new vessel formation that promote tumour cell.PDGFRB all has expression in malignant tumour, but the expression amount difference in different cancer cells.The clinical experiment data show, PDGFR is low to express the higher expresser of patient has better 3 years and get nowhere survival rate and 3 years total survival rates.Therefore, the patient carries out PDGFRB mRNA expression level and detects and to be very important before the treatment with tyrosine kinase inhibitors of accepting target PDGFRB.

(6) targeted therapy and proto-oncogene human epidermal growth factor acceptor 2 (HER-2)

HER-2 is the CerbB2 gene, is one of EGFR family member.HER-2 albumen be proto-oncogene CerbB2 (HER-2/neu) coding have an active transmembrane glycoprotein of receptor tyrosine kinase (RTK), molecular weight 185kDa, be called for short p185, belong to epidermal growth factor recipient tyrosine kinase family, can start the signal transduction system of tyrosine kinase regulatory.Trastuzumab (Herceptin, be also referred to as Herceptin, trastuzumab, Roche Group) is a kind of Humanized monoclonal antibodies at HER-2/neu proto-oncogene product, can specific combination disturb HER-2 and other ErbB family member to form heterodimer in HER2 acceptor extracellular fragment, thereby the inhibition tumor cell proliferation promotes apoptosis of tumor cells.Trastuzumab obtained the metastatic breast cancer that the drugs approved by FDA listing is used for the treatment of the HER-2 overexpression in 1998.As with one of closely-related gene of transfer prognosis of mammary cancer, it is the pathogenetic independent risk factor of mammary cancer that the crossing of HER2 acceptor expressed, and becomes one of focus of present research.

Studies show that the HER-2mRNA level is relevant with the wetting property of mammary cancer, the mammary cancer wetting property that HER-2RNA crosses expression is strong, and the disease free survival phase is short, poor prognosis.A research of North America mammary cancer cooperative groups is pointed out: during the patient with breast cancer's that determines to perform the operation HER-2 expression level, can detect this gene RNA expression amount, and can replace the IHC protein expression detection method of standard.Multinomial studies show that: the curative effect of Trastuzumab is relevant with proteic expression level with HER2mRNA.The latest data of going up report in the 44th U.S.'s Clinical Oncology annual meeting (ASCO) shows: find in the clinical study of (transitivity) mammary cancer late, the patient that HER2RNA crosses expression has long no progression of disease survival time when accepting the Trastuzumab treatment, and in the Trastuzumab treatment, progression of disease occurs and among the further women who treats of needs, use the Trastuzumab treatment still effective.(7) housekeeping gene B2M (B2M), TfR (TFRC) and TATA frame conjugated protein (TBP)

But housekeeping gene is stably express in vivo generally, but because this stably express is relative, under certain pathological state or medicining condition, the expression of the housekeeping gene that some is commonly used also can change. and the gene that is chosen in stably express in our institute's research object is made housekeeping gene, accuracy to the assurance detected result is most important. therefore, the present invention screens from the housekeeping gene commonly used of some amount, determine 3 suitable housekeeping genes, be respectively: B2M (B2M), TfR (TFRC), TATA frame conjugated protein (TBP).

Dependency based on above expression of gene level and receptor tyrosine kinase inhibitors class targeting medicament curative effect, the expert recommends the patient before accepting targeted drug, the detection of the mrna expression that should be correlated with, help the clinician to formulate the personalized medicine scheme according to patient's individual difference, to improve curative effect of medication, reduce the generation of poisonous side effect of medicine.

At present, the technology that the mRNA expression level of gene is detected mainly contains: reverse transcription quantitative PCR method, real-time fluorescence quantitative PCR method, RNA in-situ nucleic acid hybridization (RISH) etc.Wherein, RISH is mainly used in the analysis tissue or intracellular rna distributes with the expression of understanding specific gene, and its process is long, complex operation, is not suitable for clinical application; And reverse transcription quantitative PCR method and real-time fluorescence quantitative PCR method, at first, these two kinds of methods all need to carry out RNA extracting, reverse transcription, and it is big that the result of detection is influenced by the RNA degraded; Secondly, these two kinds of methods are only carried out pcr amplification by a pair of primer, are easy to generate non-specific binding, thereby cause the false positive height; Once more, these two kinds of methods generally have only a housekeeping gene to compare, and its accuracy is subjected to the influence of pathological state easily; Therefore, reverse transcription quantitative PCR method and real-time fluorescence quantitative PCR method all exist the technological deficiency that is difficult to overcome as clinical application.

Liquid-phase chip technology is utilized the carrier of polystyrene microsphere as reaction, in the manufacturing processed of microballoon, mixes the staining agent of different ratios, thereby forms the microballoon of 100 kinds of different colours codings.Thereby different microballoon covalent attachment realize the nearly detection of running simultaneously of material to be checked in 100 at the probe molecule of difference thing to be detected.Therefore, we adopt liquid-phase chip technology can detect the mRNA expression level of a plurality of genes simultaneously, have realized the high-throughput that detects, and have improved detection efficiency greatly.

Summary of the invention

One of purpose of the present invention provides receptor tyrosine kinase inhibitors class targeting medicament curative effect related gene mRNA expression level Liquid Detection chip, and this liquid-phase chip can be used for detecting the expression level of following 10 kinds of target genes: EGFR, KIT, IGF-1R, VEGFR1 (FLT1), VEGFR2 (KDR), PDGFRB, HER-2, B2M, TFRC, TBP.

The technical scheme that realizes above-mentioned purpose is as follows:

A kind of receptor tyrosine kinase inhibitors class targeting medicament curative effect related gene mRNA expression level detects liquid-phase chip, mainly includes:

(1) the support probe link coupled microballoon of modifying with amido, every support probe mainly comprise the spacerarm sequence of 5 ' end and 3 ' end with the specific sequence P1 that supports the extension probes complementary pairing, described support probe is selected from SEQ.NO.1-SEQ.NO.10, each target gene is selected a kind of microballoon for use, and every kind of microballoon has different fluorescence-encoded;

(2) connect the support extension probes of supporting probe and target gene mRNA, every support extension probes mainly comprise 5 ' end can with corresponding target gene mRNA bonded specific sequence P2, the spacerarm sequence, 3 ' end can with the P3 sequence of the specific sequence P1 complementary pairing of corresponding support probe, the specific sequence P2 of described support extension probes includes: at the EGFR gene be selected among the SEQ.NO.11-SEQ.NO.16 more than 2 or 2, at the KIT gene be selected among the SEQ.NO.17-SEQ.NO.22 more than 2 or 2, at the IGF-1R gene be selected among the SEQ.NO.23-SEQ.NO.28 more than 2 or 2, at the VEGFR1 gene be selected among the SEQ.NO.29-SEQ.NO.33 more than 2 or 2, at the VEGFR2 gene be selected among the SEQ.NO.34-SEQ.NO.38 more than 2 or 2, at the PDGFRB gene be selected among the SEQ.NO.39-SEQ.NO.43 more than 2 or 2, and/or at the HER-2 gene be selected among the SEQ.NO.44-SEQ.NO.48 more than 2 or 2; And the specific sequence P2 of described support extension probes also includes: at the B2M gene be selected among the SEQ.NO.49-SEQ.NO.53 more than 2 or 2, at the TFRC gene be selected among the SEQ.NO.54-SEQ.NO.58 more than 2 or 2 and at the TBP gene be selected among the SEQ.NO.59-SEQ.NO.63 more than 2 or 2;

(3) amplification extension probes, every amplification extension probes include 5 ' end can with target gene mRNA bonded specific sequence P4, spacerarm sequence, 3 ' terminal sequence P5,3 ' end also is modified with vitamin H; The specific sequence P4 of described amplification extension probes includes: at the EGFR gene be selected among the SEQ.NO.64-SEQ.NO.76 more than 5 or 5, at the KIT gene be selected among the SEQ.NO.77-SEQ.NO.86 more than 5 or 5, at the IGF-1R gene be selected among the SEQ.NO.87-SEQ.NO.97 more than 5 or 5, at the VEGFR1 gene be selected among the SEQ.NO.98-SEQ.NO.107 more than 5 or 5, at the VEGFR2 gene be selected among the SEQ.NO.108-SEQ.NO.117 more than 5 or 5, at the PDGFRB gene be selected among the SEQ.NO.118-SEQ.NO.127 more than 5 or 5, at the HER-2 gene be selected among the SEQ.NO.128-SEQ.NO.137 more than 5 or 5; And the specific sequence P4 of described amplification extension probes also includes: at the B2M gene be selected among the SEQ.NO.138-SEQ.NO.147 more than 5 or 5, at the TFRC gene be selected among the SEQ.NO.148-SEQ.NO.157 more than 5 or 5 and at the TBP gene be selected among the SEQ.NO.158-SEQ.NO.167 more than 5 or 5; Described P5 do not form dimer, do not have mispairing for not existing between hairpin structure, probe interior and probe, with P1, P2, P3, P4 with always all there is not the sequence of non-specific binding between the mRNA.

Perhaps, mainly comprise:

(1) the support probe link coupled microballoon of modifying with amido, every support probe mainly comprise the spacerarm sequence of 5 ' end and 3 ' end with the specific sequence P1 that supports the extension probes complementary pairing, described support probe is selected from SEQ.NO.1-SEQ.NO.10, each target gene is selected a kind of microballoon for use, and every kind of microballoon has different fluorescence-encoded;

(2) connect the support extension probes of supporting probe and target gene mRNA, every support extension probes mainly comprise 5 ' end can with corresponding target gene mRNA bonded specific sequence P2, the spacerarm sequence, 3 ' end can with the P3 sequence of the specific sequence P1 complementary pairing of corresponding support probe, the specific sequence P2 of described support extension probes includes: at the EGFR gene be selected among the SEQ.NO.11-SEQ.NO.16 more than 2 or 2, at the KIT gene be selected among the SEQ.NO.17-SEQ.NO.22 more than 2 or 2, at the IGF-1R gene be selected among the SEQ.NO.23-SEQ.NO.28 more than 2 or 2, at the VEGFR1 gene be selected among the SEQ.NO.29-SEQ.NO.33 more than 2 or 2, at the VEGFR2 gene be selected among the SEQ.NO.34-SEQ.NO.38 more than 2 or 2, at the PDGFRB gene be selected among the SEQ.NO.39-SEQ.NO.43 more than 2 or 2, and/or at the HER-2 gene be selected among the SEQ.NO.44-SEQ.NO.48 more than 2 or 2; And the specific sequence P2 of described support extension probes also includes: at the B2M gene be selected among the SEQ.NO.49-SEQ.NO.53 more than 2 or 2, at the TFRC gene be selected among the SEQ.NO.54-SEQ.NO.58 more than 2 or 2 and at the TBP gene be selected among the SEQ.NO.59-SEQ.NO.63 more than 2 or 2;

(3) amplification extension probes, every the amplification extension probes include 5 ' end can with target gene mRNA bonded specific sequence P4, spacerarm sequence, 3 ' terminal sequence P5; The specific sequence P4 of described amplification extension probes includes: at the EGFR gene be selected among the SEQ.NO.64-SEQ.NO.76 more than 5 or 5, at the KIT gene be selected among the SEQ.NO.77-SEQ.NO.86 more than 5 or 5, at the IGF-1R gene be selected among the SEQ.NO.87-SEQ.NO.97 more than 5 or 5, at the VEGFR1 gene be selected among the SEQ.NO.98-SEQ.NO.107 more than 5 or 5, at the VEGFR2 gene be selected among the SEQ.NO.108-SEQ.NO.117 more than 5 or 5, at the PDGFRB gene be selected among the SEQ.NO.118-SEQ.NO.127 more than 5 or 5, at the HER-2 gene be selected among the SEQ.NO.128-SEQ.NO.137 more than 5 or 5; And the specific sequence P4 of described amplification extension probes also includes: at the B2M gene be selected among the SEQ.NO.138-SEQ.NO.147 more than 5 or 5, at the TFRC gene be selected among the SEQ.NO.148-SEQ.NO.157 more than 5 or 5 and at the TBP gene be selected among the SEQ.NO.158-SEQ.NO.167 more than 5 or 5; Described P5 do not form dimer, do not have mispairing for not existing between hairpin structure, probe interior and probe, with P1, P2, P3, P4 with always all there is not the sequence of non-specific binding between the mRNA; With

(4) label probe, described label probe have the sequence with amplification extension probes P5 complementary pairing, and end has biotin modification.

Preferably, described receptor tyrosine kinase inhibitors class targeting medicament curative effect related gene mRNA expression level detects liquid-phase chip, also include padding sequence, comprising: at the SEQ.NO.168 of EGFR gene, SEQ.NO.169 at the KIT gene, at the IGF-1R gene be selected among the SEQ.NO.170-SEQ.NO.175 more than 1 or 1, at the VEGFR2 gene be selected among the SEQ.NO.176-SEQ.NO.181 more than 1 or 1, at the PDGFRB gene be selected among the SEQ.NO.182-SEQ.NO.187 more than 1 or 1, at the HER-2 gene be selected among the SEQ.NO.188-SEQ.NO.191 more than 1 or 1; At the B2M gene be selected among the SEQ.NO.192-SEQ.NO.199 1 or many and/or among the SEQ.NO.200-SEQ.NO.203 1 or many of being selected from of TFRC gene.

Preferably, described spacerarm sequence is 5-10 T; Described P5 consists of CCTATGCCTCCCGTGTCTA.

Another object of the present invention provides a kind of method that detects receptor tyrosine kinase inhibitors class targeting medicament curative effect related gene mRNA expression level.

Concrete technical scheme is as follows:

A kind of method that detects receptor tyrosine kinase inhibitors class targeting medicament curative effect related gene mRNA expression level is used above-mentioned liquid-phase chip, mainly may further comprise the steps:

(1) lysed sample discharges total RNA, gets the cracking mixed solution;

(2) cracking mixed solution, coupling there are microballoon, support extension probes, the amplification extension probes of supporting probe are sealed in the hybridization plate, 54 ℃ ± 1 ℃, 600rpm shakes overnight incubation, support the sequence P3 of extension probes to combine with the P1 that supports probe is complementary, support the specific sequence P2 and the target mRNA specific combination of extension probes, thereby target mRNA is attached on the microballoon; The amplification extension probes has biotin labeling, amplifies thereby the P4 of amplification extension probes and target mRNA specific combination realize the cascade of target mRNA signal;

(3) product and the Streptavidin-phycoerythrin of step (two) react;

(4) detect by fluorescence detector.

Perhaps, mainly may further comprise the steps:

(1) lysed sample discharges total RNA, gets the cracking mixed solution;

(2) cracking mixed solution, coupling there are microballoon, support extension probes, the amplification extension probes of supporting probe are sealed in the hybridization plate, 54 ℃ ± 1 ℃, 600rpm shakes overnight incubation, support the sequence P3 of extension probes to combine complementary combination with the P1 that supports probe, support the specific sequence P2 and the target mRNA specific combination of extension probes, thereby target mRNA is attached on the microballoon; The P4 and the target mRNA specific combination of amplification extension probes;

(3) add label probe again, label probe has biotin labeling, and 50 ℃ ± 1 ℃, the 600rpm concussion was hatched 1 hour, and label probe combines with the sequence P5 complementary pairing of amplification extension probes, thereby the cascade that realizes target mRNA signal is amplified;

(4) product and the Streptavidin-phycoerythrin of step (three) react;

(5) detect by fluorescence detector.

Major advantage of the present invention is:

(1) the designed various probes of the present invention can carry out hybridization under the reaction conditions of homogeneous, and do not have non-specific binding between the various probe substantially; Designed probe specificity in detection is good, signal to noise ratio is high. simultaneously, being used in combination of multiple probe makes liquid-phase chip and detection method form an intact system of detection effect. use liquid-phase chip of the present invention and detection method, the mRNA expression level is detected, need not steps such as RNA extraction, reverse transcription, PCR, detected result is subjected in the sample quality influence of RNA less, has guaranteed the accuracy of detected result; On the other hand, compare (n 〉=3) with a plurality of housekeeping genes, detected result is not subject to the influence of pathological state, and the accuracy height makes detected result more reliable.

(2) the present invention uses is that the multidigit of probe is put the amplification that mode that special pairing, cascade amplify realizes signal, rather than the method for pcr amplification, improve detection signal, realized the specificity that detects, avoided the false positive of reverse transcription PCR and real-time fluorescence quantitative PCR technology.

(3) liquid-phase chip of the present invention and detection method are applicable to that flesh tissue, paraffin embedding fixing organization, tissue slice, puncture organize the equal samples type, to mRNA detection method, the characteristics that operation is simple and easy, accuracy is high, specificity is high are arranged compared with other.

(4) the present invention can realize the detection of running simultaneously that several genes mRNA expresses in primary first-order equation, realizes that the high intension of real high-throughput detects.

Embodiment

Embodiment 1 receptor tyrosine kinase inhibitors class targeting medicament curative effect related gene mRNA expression level detects liquid phase chip reagent box, mainly includes:

One, coupling has the microballoon of support probe (SP)

Support probe to form by three parts, 5 ' end is and microballoon bonded amido end, 3 ' end is and supports extension probes bonded distinguished sequence P1, length is 16nt, the centre is the interval preface, described spacerarm is spaced apart for being used for support probe and microsphere surface, supporting extension probes and amplification extension probes inside also to have the spacerarm sequence, by between probe sequence and amino, probe interior is provided with the spacerarm sequence of suitable length, can reduce sterically hinderedly, improve the efficient of hybridization and the specificity of hybridization.The present invention supports the spacerarm of probe to be preferably 5-10 T, more preferably 8 T.Each target gene is selected a kind of microballoon for use, and selects a support probe for use, and every kind of microballoon has different fluorescence-encoded.The support probe of each target gene is as shown in table 1:

The target gene that table 1 is different and select for use microballoon numbering, support probe (SP)

Gene	Microballoon number	SP sequence (5 ' → 3 ') NH 2+poly(dT)+P1	??(P1)SEQ?ID
				??EGFR	??21	??5’NH 2-TTTTTTTT-CAAATCACAATAATCA	??1
??KIT	??14	??5’NH 2-TTTTTTTT-TCAATCAATCATCAAC	??2
				??IGF-1R	??25	??5’NH 2-TTTTTTTT-CAATTTACTCATATAC	??3
??VEGFR1	??30	??5’NH 2-TTTTTTTT-CATAATCTCATAATCC	??4
				??VEGFR2	??39	??5’NH 2-TTTTTTTT-CTCAACAATCTTTCTT	??5

Gene	Microballoon number	SP sequence (5 ' → 3 ') NH 2+poly(dT)+P1	??(P1)SEQ?ID
				??PDGFRB	??57	??5’NH 2-TTTTTTTT-TCTAACTTCACTATTA	??6
??HER-2	??13	??5’NH 2-TTTTTTTT-CATATTACATTCACAT	??7
				??B2M	??22	??5’NH 2-TTTTTTTT-CTTTCTTTAATCTCAA	??8
??TFRC	??61	??5’NH 2-TTTTTTTT-CATTCAAATCTCAACT	??9
				??TBP	??33	??5’NH 2-TTTTTTTT-CAATTACTTCAAATCT	??10

All probes are synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Synthetic is supported probe sterilization ddH ₂O is made into the stock solution of 100nmol/ml.The process of microballoon bag quilt is as follows:

Get 5 * 10 respectively ⁶The carboxylated microballoon of individual above-mentioned numbering (available from Luminex company) is suspended in the MES solution of 50ul 0.1mol/L (pH4.5), adds 10ul synthetic capture probe (100nmol/ml).EDC (N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide) (available from the Pierce Chemical company) working fluid of preparation 10ng/ml.Add the EDC working fluid of 2.5ul in the microballoon suspension, constant temperature was hatched 30 minutes, added the EDC working fluid of 2.5ul again, and constant temperature was hatched 30 minutes again.After reaction finished, the Tween-20 washing with 0.02% was once washed once with 0.1% SDS liquid again.Support the microballoon of probe to be resuspended in the Tris-EDTA solution [10mmol/L Tris (pH8.0), 1mmol/LEDTA] of 100ul being coated with after the washing, 2-8 ℃ keeps in Dark Place.

Two, with the support extension probes (SE) of target gene mRNA specific combination, amplification extension probes (AE)

1) supports that extension probes (SE) is to connect the sequence of supporting between probe (SP) and the target gene mRNA, SE is made up of three parts, 5 ' end be can with target gene bonded distinguished sequence P2, length is between 17nt to 27nt, 3 ' end is to pass through base complementrity bonded distinguished sequence P3 with supporting probe 5 ' end, the centre is an intervening sequence, and support extension probes spacerarm of the present invention is preferably 5-10 T, more preferably 5 T.Each target gene designs 5 to 6 respectively and supports extension probes, to improve the specificity that detects.During use, at every kind of target gene, select to support more than 2 or 2 extension probes can finish detection, specificity and stability all fine (experimental data omission) are preferable over and use 5 or 6 to support extension probes, so that specificity reaches best.Every probe after synthetic is mixed with the stock solution of 100pmol/mL respectively with 10mmol/L Tris Buffer.The support extension probes (SE) of target gene sees Table 2.

The support extension probes (SE) of table 2 target gene

2) amplification extension probes (AE) is the sequence between linking objective gene mRNA and the signal detection component, AE is made up of three parts, 5 ' end be can with target gene mRNA bonded specific sequence P4,3 ' end be can with the sequence P5 of label probe complementary pairing, (when not using label probe to detect, then 3 ' end also is modified with vitamin H), the centre is the spacerarm sequence (amplification extension probes spacerarm of the present invention is preferably 5-10 T, more preferably 5 T) of 5 oligonucleotide T.The amplification extension probes (AE) of target gene sees Table 3.The design of described P5 is according to the common practise of the probe design of this area, it is not for existing inner hairpin structure, do not form dimer between probe interior and probe, do not have mispairing, and all do not have the sequence of non-specific binding between P1, P2, P3, P4 and the total mRNA, it is CCTATGCCTCCCGTGTCTA that the present invention is preferably based composition.

Each target gene designs 10 to 13 amplification extension probes respectively, amplifies thereby detection signal is carried out cascade.Every probe after synthetic is mixed with the stock solution of 100pmol/mL respectively with 10mmol/L Tris Buffer.During use, at every kind of target gene, the extension probes of selecting to increase more than 5 or 5 can be finished detection, and specificity and stability all fine (experimental data omission) are preferable over and use 10 or 12 amplification extension probes, so that specificity reaches best.

The amplification extension probes (AE) of table 3 target gene

3) padding sequence (FS) will not seal with the zone of SE, AE specific combination among the target gene mRNA, with the non-specific binding of vitamin H or label probe in the minimizing subsequent reactions, thereby reduce detection background.The Tm value of padding sequence should be approaching with SE, AE, as shown in table 4.

The padding sequence of table 4 target gene (FS)

Three, label probe (LP)

Label probe (LP) is made up of two portions, its 3 ' end be can with the complementary bonded sequence of amplification extension probes sequence P5,5 ' end has biotin labeling, amplifies by combining the cascade that realizes target mRNA signal with the amplification extension probes.

Table 5 label probe

Embodiment 2 utilization receptor tyrosine kinase inhibitors class targeted drug related gene mRNA expressive level detection Luminexes are to the detection of clinical sample

The prescription of described various solution is as follows:

One, lysed sample discharges total RNA

1. FFPE tumor tissue section is scraped from slide glass, put into the 1.5ml centrifuge tube, add 300ul homogenate buffer and 3ul Proteinase K (50ug/ul), mix, 65 ℃ of digestion 2h.

2. the sample of digestion behind the 2h, under the room temperature 13, centrifugal 5 minutes of 000rpm, clear soln is transferred in the new 1.5ml centrifuge tube standby in the middle of drawing. (still do not clarify as solution, then repeat this step 1～2 time .)

Two, target mRNA is fixed on the microballoon by combining with probe specificity

1. take out the probe working fluid and melt,, place on ice at once then 95 ℃ of sex change 5 minutes in room temperature.

2. according to the form below preparation working fluid mixes

3. the above-mentioned working fluid branch for preparing is filled on the hybridization plate 60ul/ hole.Then the above-mentioned sample of handling well is added respectively in the hybridization plate with the 40ul/ hole; Blank adds homogenate buffer 40ul/ hole.Sealing hybridization plate, 54 ℃ ± 1 ℃, 600rpm shakes overnight incubation (16-22h).

4. use washing buffer liquid wetting filter plate 1 minute, remove lavation buffer solution.

5. will hybridize plate and take out, centrifugal 1 minute of 500 * g all is transferred to reaction solution in the filter plate.

6. remove solution, with 200ul lavation buffer solution washing 3 times.

Three, by hybridization target RNA signal is amplified

1. add label probe working fluid 100ul/ hole.50 ℃ ± 1 ℃, the 600rpm concussion was hatched 1 hour.

2. remove solution, with 200ul washings washing 2 times.

Four, combine with SA-PE, and on the liquid-phase chip reading apparatus, detect

1. add SA-PE working fluid 100ul/ hole, 600rpm concussion incubated at room 30min.

2. remove solution, with 200ul SA-PE washings washing 2 times.

3. add SA-PE washings 130ul/ hole.Room temperature, the 600rpm concussion was hatched 2 to 5 minutes.

4. reading of data on the liquid-phase chip instrument.

Five, data analysis

In 10 target genes, 7 of marker gene are respectively: EGFR, KIT, IGF-1R, VEGFR1, VEGFR2, PDGFRB, HER-2;

3 of housekeeping genes are respectively: B2M, TFRC, TBP;

The fluorescent value that reads is carried out homogenization in accordance with the following methods to be handled:

Step 1: obtain raw data (MFI value)

Step 2: the MFI of sample deducts the MFI=net MFI of blank well N

Step 3: the net MFI value of three housekeeping genes of each sample is got geometrical mean respectively, obtains corresponding G1, G2, G3......Gn

Step 4: the net MFI of each sample target gene mRNA obtains corresponding N n.Nn divided by the Gn of correspondence and is and gets rid of applied sample amount difference, can carry out relative expression quantity numerical value relatively at sample room.

In the present embodiment 10 routine samples are detected, detected result is as follows:

Table 6 blank well N and 10 routine sample raw data (MFI value)

??Sample	??EGFR	??KIT	??IGF1R	??VEGFR1	??VEGFR2	??PDGFRB	??HER2	?B2M	??TFRC	??TBP
											??N	??4	??6	??7	??6	??5	??5	??5	?4	??7	??7
??1	??1462	??204	??1016	??217	??419	??225	??1035	?14638	??10139	??780
											??2	??665	??472	??917	??196	??502	??149	??1841	?18907	??9417	??729
??3	??4827	??272	??446	??179	??388	??166	??1174	?23707	??17517	??1158
											??4	??1838	??597	??917	??181	??869	??272	??947	?21064	??4219	??588
??5	??276	??1517	??855	??241	??383	??198	??331	?21662	??4286	??1368
											??6	??2373	??189	??1998	??116	??236	??147	??625	?8626	??7700	??499
??7	??2692	??161	??1132	??110	??130	??203	??620	?10792	??3494	??331
											??8	??1706	??983	??1666	??622	??1334	??1423	??2247	?21294	??3882	??874
??9	??3267	??507	??1124	??313	??521	??580	??1236	?25566	??8299	??1174
											??10	??4152	??1943	??1114	??99	??6113	??107	??3934	?17067	??2016	??714

The relative expression quantity of table 7 10 routine each gene of sample

??Sample	??EGFR	??KIT	??IGF1R	??VEGFR1	??VEGFR2	??PDGFRB	??HER2
								??1	??0.300	??0.041	??0.208	??0.044	??0.085	??0.045	??0.212
??2	??0.131	??0.092	??0.180	??0.038	??0.099	??0.028	??0.364
								??3	??0.617	??0.034	??0.056	??0.022	??0.049	??0.021	??0.150
??4	??0.493	??0.159	??0.245	??0.047	??0.232	??0.072	??0.253
								??5	??0.054	??0.301	??0.169	??0.047	??0.075	??0.038	??0.065
??6	??0.741	??0.057	??0.623	??0.035	??0.072	??0.044	??0.194

??Sample	??EGFR	??KIT	??IGF1R	??VEGFR1	??VEGFR2	??PDGFRB	??HER2
								??7	??1.167	??0.067	??0.489	??0.045	??0.055	??0.086	??0.267
??8	??0.410	??0.235	??0.400	??0.148	??0.320	??0.342	??0.540
								??9	??0.520	??0.080	??0.178	??0.049	??0.082	??0.092	??0.196
??10	??1.433	??0.669	??0.383	??0.032	??2.110	??0.035	??1.357

The liquid-phase chip of embodiment 3 different interval arms is to the detection of receptor tyrosine kinase inhibitors class targeting medicament curative effect related gene mRNA expression level

One, the design (selection of spacerarm) of liquid-phase chip preparation is an example with the support probe that B2M, EGFR, IGF-1R detect liquid-phase chip, selects different spacerarms respectively for use, and specific design is as shown in table 8.Probe SP, SE and AE synthetic, SP sequence bag is described like embodiment 1 and embodiment 2 by microballoon, detection method.

Table 8 spacerarm and length thereof

The spacerarm kind	Length	Experimental group
			??poly(dT)	??8	??1
??poly(dA)	??8	??2
			??(CH2)n	??15	??3
??poly(TTG)	??3	??4

Two, sample detection

Adopt the liquid-phase chip of above-mentioned design preparation, sample 11-15 detected, detected result following (data are for detecting fluorescent value in the table) by embodiment 2 described testing processes and method:

Table 9a sample B2M detected result (detect fluorescent value＜MFI value 〉)

Table 9b sample EGFR detected result (detect fluorescent value＜MFI value 〉)

Table 9c sample IGF-1R detected result (detect fluorescent value＜MFI value 〉)

The detection fluorescent value of 4 groups of designs is carried out statistical analysis, prove that the detection effect of 4 groups of designs does not have difference.Therefore, the design of these 4 kinds of spacerarms is equivalent.

Other is at the liquid-phase chip of supporting extension probes, intervening sequence that the amplification extension probes is inner different, and its result is still reliable and stable, and concrete data are omitted.

Embodiment 4: the utilization of label probe

The design (signal detection component) of liquid-phase chip preparation

Described signal detection component has two kinds of selections, 1) 3 ' end of amplification extension probes 3 ' terminal sequence P5 has biotin labeling; 2) amplification extension probes 3 ' terminal sequence P5 combines by the base complementrity pairing with label probe (LP), and 5 ' of label probe end has biotin labeling simultaneously.These two kinds of signal detection components can realize that all signal amplifies, and detects normal signal.Wherein, the vitamin H activity of applying marking probe is more stable, and effect is more excellent.

With detection B2M, the EGFR of described liquid-phase chip, two kinds of signal detection components of IGF-1R gene is example, and specific design is as shown in table 10.Be consisting of of described liquid-phase chip:

Experimental group 1: support probe link coupled microballoon, support extension probes, padding sequence with embodiment 1, the amplification extension probes has biotin labeling; There is not label probe;

Experimental group 2: support probe link coupled microballoon, support extension probes, padding sequence, label probe with embodiment 1, the amplification extension probes does not have biotin labeling.

Table 10 signal detection component

Two, sample detection

Adopt the liquid-phase chip of above-mentioned design preparation, sample 16-20 detected, detected result following (data are for detecting fluorescent value in the table) by embodiment 2 described testing processes and method:

Table 11 sample detection result (detect fluorescent value＜MFI value 〉)

The detection fluorescent value of two groups of designs is carried out statistical analysis, prove that the detected result of two groups of designs does not have difference, therefore, these two kinds of signal detection components are equivalent to the detection of signal.Wherein, the signal detection component of applying marking probe, because the activity of its vitamin H is more stable, effect is more excellent.

More than be at the specifying of possible embodiments of the present invention, but this embodiment is not in order to limiting claim of the present invention, does not allly break away from the equivalence that skill spirit of the present invention does and implement or change, all should be contained in the claim of the present invention.

Sequence table

＜110〉Guangzhou Yishan Biotechnology Co., Ltd.

＜120〉the targeting medicament curative effect related gene mRNA expression level detects liquid-phase chip and detection method

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<212>DNA

＜213〉artificial sequence

<400>92

ccctggtccc?ccagcaag????18

<210>93

<211>18

<212>DNA

＜213〉artificial sequence

<400>93

agggaaagtg?ggccagca????18

<210>94

<211>21

<212>DNA

＜213〉artificial sequence

<400>94

aacaagacaa?gacccctggg?c????????21

<210>95

<211>25

<212>DNA

＜213〉artificial sequence

<400>95

tgctgtcatc?tctctggtga?gaagt????25

<210>96

<211>26

<212>DNA

＜213〉artificial sequence

<400>96

cgtatcccag?ctttgttctt?actcct??26

<210>97

<211>23

<212>DNA

＜213〉artificial sequence

<400>97

ttaaagggaa?cacgctatgg?gat????23

<210>98

<211>17

<212>DNA

＜213〉artificial sequence

<400>98

gagcagcgcg?cacagca??????????????17

<210>99

<211>25

<212>DNA

＜213〉artificial sequence

<400>99

ctagatcctg?tgagaagcag?acagc????25

<210>100

<211>20

<212>DNA

＜213〉artificial sequence

<400>100

tgcttgcatg?atgtgctggg??????????20

<210>101

<211>22

<212>DNA

＜213〉artificial sequence

<400>101

gcattggaga?tgcagtgtct?gg??????22

<210>102

<211>24

<212>DNA

＜213〉artificial sequence

<400>102

ctttccttac?tcaccatttc?aggc????24

<210>103

<211>25

<212>DNA

＜213〉artificial sequence

<400>103

gcagatttag?ttatgctcag?ccttt????25

<210>104

<211>25

<212>DNA

＜213〉artificial sequence

<400>104

gcttgagctg?tgttcaaggt?taaag????25

<210>105

<211>23

<212>DNA

＜213〉artificial sequence

<400>105

gcagctgtag?aagccagtgt?ggt????23

<210>106

<211>25

<212>DNA

＜213〉artificial sequence

<400>106

tcggggattt?cactgtacat?ctcta????25

<210>107

<211>23

<212>DNA

＜213〉artificial sequence

<400>107

cgagctccct?tccttcagtc?atg????23

<210>108

<211>25

<212>DNA

＜213〉artificial sequence

<400>108

ccacttggaa?gctgtaacag?atgag??25

<210>109

<211>22

<212>DNA

＜213〉artificial sequence

<400>109

gatgccaaga?actccatgcc?ct????22

<210>110

<211>23

<212>DNA

＜213〉artificial sequence

<400>110

cgataagagg?atatttcgtg?ccg????23

<210>111

<211>25

<212>DNA

＜213〉artificial sequence

<400>111

gccaaagtca?cagattttaa?ccacg????25

<210>112

<211>25

<212>DNA

＜213〉artificial sequence

<400>112

cgagcatctc?cttttctgac?ataat????25

<210>113

<211>20

<212>DNA

＜213〉artificial sequence

<400>113

gggccatcca?tttcaaaggg??????????20

<210>114

<211>23

<212>DNA

＜213〉artificial sequence

<400>114

cccacagcaa?aacaccaaaa?gac??????23

<210>115

<211>25

<212>DNA

＜213〉artificial sequence

<400>115

ggatatggag?aagcacctaa?ggaaa????25

<210>116

<211>24

<212>DNA

＜213〉artificial sequence

<400>116

ggtgtagtat?aatcaggggc?cctc?????24

<210>117

<211>23

<212>DNA

＜213〉artificial sequence

<400>117

tcccaaatgt?tccaccaact?ctg??????23

<210>118

<211>21

<212>DNA

＜213〉artificial sequence

<400>118

cgggggtatg?tccactcgaa?g????????21

<210>119

<211>19

<212>DNA

＜213〉artificial sequence

<400>119

accagccgcc?cactttctt??????????19

<210>120

<211>21

<212>DNA

＜213〉artificial sequence

<400>120

tggagcggat?gtggtaaggc?a????21

<210>121

<211>21

<212>DNA

＜213〉artificial sequence

<400>121

tctaactcgg?cactggggat?g????21

<210>122

<211>24

<212>DNA

＜213〉artificial sequence

<400>122

tgatggtcat?tcacactctc?cgtc????24

<210>123

<211>22

<212>DNA

＜213〉artificial sequence

<400>123

cggtgatgtt?gatggccttt?tc????22

<210>124

<211>20

<212>DNA

＜213〉artificial sequence

<400>124

tagtgtgccc?acctctccca???????20

<210>125

<211>22

<212>DNA

＜213〉artificial sequence

<400>125

gcggttgtct?ttgaaccaca?gg????22

<210>126

<211>22

<212>DNA

＜213〉artificial sequence

<400>126

tcagctctga?cacataccgg?gt????22

<210>127

<211>22

<212>DNA

＜213〉artificial sequence

<400>127

tagctggaag?gagagctgga?cc????22

<210>128

<211>25

<212>DNA

＜213〉artificial sequence

<400>128

gaaaggtagt?tgtagggaca?ggcag????25

<210>129

<211>19

<212>DNA

＜213〉artificial sequence

<400>129

agggtgcagg?atcccacgt??????????19

<210>130

<211>23

<212>DNA

＜213〉artificial sequence

<400>130

ctgtgttcca?tcctctgctg?tca????23

<210>131

<211>21

<212>DNA

＜213〉artificial sequence

<400>131

gggcttgctg?cacttctcac?a????21

<210>132

<211>21

<212>DNA

＜213〉artificial sequence

<400>132

cacctctcgc?aagtgctcca?t????21

<210>133

<211>24

<212>DNA

＜213〉artificial sequence

<400>133

cctggatatt?ggcactggta?actg????24

<210>134

<211>20

<212>DNA

＜213〉artificial sequence

<400>134

gtccccatca?aagctctccg????????20

<210>135

<211>18

<212>DNA

＜213〉artificial sequence

<400>135

gagcggggca?gtgttgga??????????18

<210>136

<211>23

<212>DNA

＜213〉artificial sequence

<400>136

cggccatgct?gagatgtata?ggt????23

<210>137

<211>20

<212>DNA

＜213〉artificial sequence

<400>137

cagcgagtag?gcgccattgt????????20

<210>138

<211>17

<212>DNA

＜213〉artificial sequence

<400>138

ttcaggaatg?cccgcca???????????17

<210>139

<211>23

<212>DNA

＜213〉artificial sequence

<400>139

ccaggccaga?aagagagagt?agc????23

<210>140

<211>23

<212>DNA

＜213〉artificial sequence

<400>140

cctgaatctt?tggagtacgc?tgg????23

<210>141

<211>24

<212>DNA

＜213〉artificial sequence

<400>141

ggatgaaacc?cagacacata?gcaa????24

<210>142

<211>24

<212>DNA

＜213〉artificial sequence

<400>142

agtgggggtg?aattcagtgt?agta????24

<210>143

<211>23

<212>DNA

＜213〉artificial sequence

<400>143

tcacacggca?ggcatactca?tct????23

<210>144

<211>22

<212>DNA

＜213〉artificial sequence

<400>144

gctgcttaca?tgtctcgatc?cc????22

<210>145

<211>20

<212>DNA

＜213〉artificial sequence

<400>145

tgcggcatct?tcaaacctcc??????????20

<210>146

<211>25

<212>DNA

＜213〉artificial sequence

<400>146

gcaacctgct?cagatacatc?aaaca????25

<210>147

<211>23

<212>DNA

＜213〉artificial sequence

<400>147

cctctaagtt?gccagccctc?cta??????23

<210>148

<211>25

<212>DNA

＜213〉artificial sequence

<400>148

atagtcccat?agcagatact?tccac????25

<210>149

<211>25

<212>DNA

＜213〉artificial sequence

<400>149

caatcaagaa?aaagacgatc?acagc????25

<210>150

<211>23

<212>DNA

＜213〉artificial sequence

<400>150

ctcagttttt?ggttctaccc?ctt????23

<210>151

<211>21

<212>DNA

＜213〉artificial sequence

<400>151

ctcctggctc?ctccctcact?g????21

<210>152

<211>19

<212>DNA

＜213〉artificial sequence

<400>152

cgacgtgctg?cagggaagt???????19

<210>153

<211>22

<212>DNA

＜213〉artificial sequence

<400>153

gctggtgaag?tctgtgctgt?cc????22

<210>154

<211>22

<212>DNA

＜213〉artificial sequence

<400>154

ttttcattca?gcagcttgat?gg????22

<210>155

<211>22

<212>DNA

＜213〉artificial sequence

<400>155

cgagttttga?gcgctgtctt?tg????22

<210>156

<211>24

<212>DNA

＜213〉artificial sequence

<400>156

caggattctc?caccaggtaa?acaa????24

<210>157

<211>23

<212>DNA

＜213〉artificial sequence

<400>157

gttgcagcct?tactatacgc?cac????23

<210>158

<211>19

<212>DNA

＜213〉artificial sequence

<400>158

gcagctgcgg?tacaatccc???????????19

<210>159

<211>25

<212>DNA

＜213〉artificial sequence

<400>159

tacaaccaag?attcactgtg?gatac????25

<210>160

<211>22

<212>DNA

＜213〉artificial sequence

<400>160

gattatattc?ggcgtttcgg?gc???????22

<210>161

<211>21

<212>DNA

＜213〉artificial sequence

<400>161

atgattaccg?cagcaaaccg?c???????21

<210>162

<211>22

<212>DNA

＜213〉artificial sequence

<400>162

gcacaccatt?ttcccagaac?tg????22

<210>163

<211>23

<212>DNA

＜213〉artificial sequence

<400>163

ctgttcttca?ctcttggctc?ctg????23

<210>164

<211>24

<212>DNA

＜213〉artificial sequence

<400>164

acccaacttc?tgtacaactc?tagc????24

<210>165

<211>25

<212>DNA

＜213〉artificial sequence

<400>165

tcttgaagtc?caagaactta?gctgg????25

<210>166

<211>22

<212>DNA

＜213〉artificial sequence

<400>166

gggtgagcac?aaggccttct?aa????22

<210>167

<211>26

<212>DNA

＜213〉artificial sequence

<400>167

caggaaataa?ctctggctca?taacta????26

<210>168

<211>20

<212>DNA

＜213〉artificial sequence

<400>168

ccgttacaca?ctttgcggca???????????20

<210>169

<211>25

<212>DNA

＜213〉artificial sequence

<400>169

cataattgtt?tccatttatc?tcctc????25

<210>170

<211>18

<212>DNA

＜213〉artificial sequence

<400>170

ggtccggtga?ggggggtg???????????18

<210>171

<211>25

<212>DNA

＜213〉artificial sequence

<400>171

catttctatc?ccagaagcaa?ctctt????25

<210>172

<211>26

<212>DNA

＜213〉artificial sequence

<400>172

tttctgtgtt?gaatcttcag?tcacaa????26

<210>173

<211>26

<212>DNA

＜213〉artificial sequence

<400>173

agactagaga?aagcagtggg?acaaac????26

<210>174

<211>30

<212>DNA

＜213〉artificial sequence

<400>174

cacaaaataa?attaaaagaa?aactcctaca?30

<210>175

<211>27

<212>DNA

＜213〉artificial sequence

<400>175

cttttctaca?gtgattgaaa?ctggtaa????27

<210>176

<211>22

<212>DNA

＜213〉artificial sequence

<400>176

attcttcatc?aatctttacc?cc????????22

<210>177

<211>23

<212>DNA

＜213〉artificial sequence

<400>177

agcagtccag?catggtctgg?tac???????23

<210>178

<211>20

<212>DNA

＜213〉artificial sequence

<400>178

tgggtctctg?actgggctcc???????????20

<210>179

<211>21

<212>DNA

＜213〉artificial sequence

<400>179

cctgctgagc?attagcttgc?a?????????21

<210>180

<211>26

<212>DNA

＜213〉artificial sequence

<400>180

aagtctctga?tatcggaaga?acaatg????26

<210>181

<211>25

<212>DNA

＜213〉artificial sequence

<400>181

agagagtcca?gaatcctctt?ccatg????25

<210>182

<211>18

<212>DNA

＜213〉artificial sequence

<400>182

gtgtccggct?ccgatgca????????????18

<210>183

<211>22

<212>DNA

＜213〉artificial sequence

<400>183

ggtgggtagg?cctcgaacac?ta????????22

<210>184

<211>18

<212>DNA

＜213〉artificial sequence

<400>184

acgttgcgcg?tggacagg????????18

<210>185

<211>18

<212>DNA

＜213〉artificial sequence

<400>185

tctgccacct?tcacgcga????????18

<210>186

<211>19

<212>DNA

＜213〉artificial sequence

<400>186

ggaaggcccg?catggtgta???????19

<210>187

<211>21

<212>DNA

＜213〉artificial sequence

<400>187

agcactcgga?cagggacatt?g????21

<210>188

<211>21

<212>DNA

＜213〉artificial sequence

<400>188

ggttctggaa?gacgctgagg?t????21

<210>189

<211>22

<212>DNA

＜213〉artificial sequence

<400>189

gaattcgtcc?ccggattact?tg????22

<210>190

<211>18

<212>DNA

＜213〉artificial sequence

<400>190

cccagccagc?tgatgccc?????????18

<210>191

<211>20

<212>DNA

＜213〉artificial sequence

<400>191

tccactgccc?agttccctca???????20

<210>192

<211>17

<212>DNA

＜213〉artificial sequence

<400>192

cggcccgaat?gctgtca???????????17

<210>193

<211>22

<212>DNA

＜213〉artificial sequence

<400>193

cagtaagtca?acttcaatgt?cg?????22

<210>194

<211>23

<212>DNA

＜213〉artificial sequence

<400>194

ctttttcaat?tctctctcca?ttc????23

<210>195

<211>20

<212>DNA

＜213〉artificial sequence

<400>195

agagatagaa?agaccagtcc????????20

<210>196

<211>23

<212>DNA

＜213〉artificial sequence

<400>196

agaatttgga?attcatccaa?tcc????23

<210>197

<211>23

<212>DNA

＜213〉artificial sequence

<400>197

catatcaata?ttaaaaagca?agc????23

<210>198

<211>23

<212>DNA

＜213〉artificial sequence

<400>198

ctacattttg?tgcataaagt?gta????23

<210>199

<211>23

<212>DNA

＜213〉artificial sequence

<400>199

gaagatcatg?tccatgttaa?cat????23

<210>200

<211>23

<212>DNA

＜213〉artificial sequence

<400>200

cgcaagattt?tcatcttttt?gag????23

<210>201

<211>23

<212>DNA

＜213〉artificial sequence

<400>201

cacgaaattg?attttcaaca?tac????23

<210>202

<211>22

<212>DNA

＜213〉artificial sequence

<400>202

ctgaatctta?acaaaatgtt?ga?????22

<210>203

<211>23

<212>DNA

＜213〉artificial sequence

<400>203

ctaccgttct?tatcaactat?gat????23

<210>204

<211>19

<212>DNA

＜213〉artificial sequence

<400>204

tagacacggg?aggcatagg?????????19

Claims (10)

1. a receptor tyrosine kinase inhibitors class targeting medicament curative effect related gene mRNA expression level detects liquid-phase chip, it is characterized in that, mainly includes:

(1) the support probe link coupled microballoon of modifying with amido, every support probe mainly comprise the spacerarm sequence of 5 ' end and 3 ' end with the specific sequence P1 that supports the extension probes complementary pairing, described support probe is selected from SEQ.NO.1-SEQ.NO.10, each target gene is selected a kind of microballoon for use, and every kind of microballoon has different fluorescence-encoded;

(2) connect the support extension probes of supporting probe and target gene mRNA, every support extension probes mainly comprise 5 ' end can with corresponding target gene mRNA bonded specific sequence P2, the spacerarm sequence, 3 ' end can with the P3 sequence of the specific sequence P1 complementary pairing of corresponding support probe, the specific sequence P2 of described support extension probes includes: at the EGFR gene be selected among the SEQ.NO.11-SEQ.NO.16 more than 2 or 2, at the KIT gene be selected among the SEQ.NO.17-SEQ.NO.22 more than 2 or 2, at the IGF-1R gene be selected among the SEQ.NO.23-SEQ.NO.28 more than 2 or 2, at the VEGFR1 gene be selected among the SEQ.NO.29-SEQ.NO.33 more than 2 or 2, at the VEGFR2 gene be selected among the SEQ.NO.34-SEQ.NO.38 more than 2 or 2, at the PDGFRB gene be selected among the SEQ.NO.39-SEQ.NO.43 more than 2 or 2, and/or at the HER-2 gene be selected among the SEQ.NO.44-SEQ.NO.48 more than 2 or 2; And the specific sequence P2 of described support extension probes also includes: at the B2M gene be selected among the SEQ.NO.49-SEQ.NO.53 more than 2 or 2, at the TFRC gene be selected among the SEQ.NO.54-SEQ.NO.58 more than 2 or 2 and at the TBP gene be selected among the SEQ.NO.59-SEQ.NO.63 more than 2 or 2;

(3) amplification extension probes, every amplification extension probes include 5 ' end can with target gene mRNA bonded specific sequence P4, spacerarm sequence, 3 ' terminal sequence P5,3 ' end also is modified with vitamin H; The specific sequence P4 of described amplification extension probes includes: at the EGFR gene be selected among the SEQ.NO.64-SEQ.NO.76 more than 5 or 5, at the KIT gene be selected among the SEQ.NO.77-SEQ.NO.86 more than 5 or 5, at the IGF-1R gene be selected among the SEQ.NO.87-SEQ.NO.97 more than 5 or 5, at the VEGFR1 gene be selected among the SEQ.NO.98-SEQ.NO.107 more than 5 or 5, at the VEGFR2 gene be selected among the SEQ.NO.108-SEQ.NO.117 more than 5 or 5, at the PDGFRB gene be selected among the SEQ.NO.118-SEQ.NO.127 more than 5 or 5, at the HER-2 gene be selected among the SEQ.NO.128-SEQ.NO.137 more than 5 or 5; And the specific sequence P4 of described amplification extension probes also includes: at the B2M gene be selected among the SEQ.NO.138-SEQ.NO.147 more than 5 or 5, at the TFRC gene be selected among the SEQ.NO.148-SEQ.NO.157 more than 5 or 5 and at the TBP gene be selected among the SEQ.NO.158-SEQ.NO.167 more than 5 or 5; Described P5 do not form dimer, do not have mispairing for not existing between hairpin structure, probe interior and probe, with P1, P2, P3, P4 with always all there is not the sequence of non-specific binding between the mRNA.

2. receptor tyrosine kinase inhibitors class targeting medicament curative effect related gene mRNA expression level according to claim 1 detects liquid-phase chip, it is characterized in that, also include padding sequence, comprising: at the SEQ.NO.168 of EGFR gene, SEQ.NO.169 at the KIT gene, at the IGF-1R gene be selected among the SEQ.NO.170-SEQ.NO.175 more than 1 or 1, at the VEGFR2 gene be selected among the SEQ.NO.176-SEQ.NO.181 more than 1 or 1, at the PDGFRB gene be selected among the SEQ.NO.182-SEQ.NO.187 more than 1 or 1, at the HER-2 gene be selected among the SEQ.NO.188-SEQ.NO.191 more than 1 or 1; At the B2M gene be selected among the SEQ.NO.192-SEQ.NO.199 1 or many and/or among the SEQ.NO.200-SEQ.NO.203 1 or many of being selected from of TFRC gene.

3. receptor tyrosine kinase inhibitors class targeting medicament curative effect related gene mRNA expression level according to claim 1 and 2 detects liquid-phase chip, it is characterized in that described spacerarm sequence is 5-10 T.

4. receptor tyrosine kinase inhibitors class targeting medicament curative effect related gene mRNA expression level according to claim 1 detects liquid-phase chip, it is characterized in that, described P5 consists of CCTATGCCTCCCGTGTCTA.

5. a receptor tyrosine kinase inhibitors class targeting medicament curative effect related gene mRNA expression level detects liquid-phase chip, it is characterized in that, mainly includes:

(1) the support probe link coupled microballoon of modifying with amido, every support probe mainly comprise the spacerarm sequence of 5 ' end and 3 ' end with the specific sequence P1 that supports the extension probes complementary pairing, described support probe is selected from SEQ.NO.1-SEQ.NO.10, each target gene is selected a kind of microballoon for use, and every kind of microballoon has different fluorescence-encoded;

(2) connect the support extension probes of supporting probe and target gene mRNA, every support extension probes mainly comprise 5 ' end can with corresponding target gene mRNA bonded specific sequence P2, the spacerarm sequence, 3 ' end can with the P3 sequence of the specific sequence P1 complementary pairing of corresponding support probe, the specific sequence P2 of described support extension probes includes: at the EGFR gene be selected among the SEQ.NO.11-SEQ.NO.16 more than 2 or 2, at the KIT gene be selected among the SEQ.NO.17-SEQ.NO.22 more than 2 or 2, at the IGF-1R gene be selected among the SEQ.NO.23-SEQ.NO.28 more than 2 or 2, at the VEGFR1 gene be selected among the SEQ.NO.29-SEQ.NO.33 more than 2 or 2, at the VEGFR2 gene be selected among the SEQ.NO.34-SEQ.NO.38 more than 2 or 2, at the PDGFRB gene be selected among the SEQ.NO.39-SEQ.NO.43 more than 2 or 2, and/or at the HER-2 gene be selected among the SEQ.NO.44-SEQ.NO.48 more than 2 or 2; And the specific sequence P2 of described support extension probes also includes: at the B2M gene be selected among the SEQ.NO.49-SEQ.NO.53 more than 2 or 2, at the TFRC gene be selected among the SEQ.NO.54-SEQ.NO.58 more than 2 or 2 and at the TBP gene be selected among the SEQ.NO.59-SEQ.NO.63 more than 2 or 2;

(3) amplification extension probes, every the amplification extension probes include 5 ' end can with target gene mRNA bonded specific sequence P4, spacerarm sequence, 3 ' terminal sequence P5; The specific sequence P4 of described amplification extension probes includes: at the EGFR gene be selected among the SEQ.NO.64-SEQ.NO.76 more than 5 or 5, at the KIT gene be selected among the SEQ.NO.77-SEQ.NO.86 more than 5 or 5, at the IGF-1R gene be selected among the SEQ.NO.87-SEQ.NO.97 more than 5 or 5, at the VEGFR1 gene be selected among the SEQ.NO.98-SEQ.NO.107 more than 5 or 5, at the VEGFR2 gene be selected among the SEQ.NO.108-SEQ.NO.117 more than 5 or 5, at the PDGFRB gene be selected among the SEQ.NO.118-SEQ.NO.127 more than 5 or 5, at the HER-2 gene be selected among the SEQ.NO.128-SEQ.NO.137 more than 5 or 5; And the specific sequence P4 of described amplification extension probes also includes: at the B2M gene be selected among the SEQ.NO.138-SEQ.NO.147 more than 5 or 5, at the TFRC gene be selected among the SEQ.NO.148-SEQ.NO.157 more than 5 or 5 and at the TBP gene be selected among the SEQ.NO.158-SEQ.NO.167 more than 5 or 5; Described P5 do not form dimer, do not have mispairing for not existing between hairpin structure, probe interior and probe, with P1, P2, P3, P4 with always all there is not the sequence of non-specific binding between the mRNA; With

(4) label probe, described label probe have the sequence with amplification extension probes P5 complementary pairing, and end has biotin modification.

6. receptor tyrosine kinase inhibitors class targeting medicament curative effect related gene mRNA expression level according to claim 5 detects liquid-phase chip, it is characterized in that, also include padding sequence, comprising: at the SEQ.NO.168 of EGFR gene, SEQ.NO.169 at the KIT gene, at the IGF-1R gene be selected among the SEQ.NO.170-SEQ.NO.175 more than 1 or 1, at the VEGFR2 gene be selected among the SEQ.NO.176-SEQ.NO.181 more than 1 or 1, at the PDGFRB gene be selected among the SEQ.NO.182-SEQ.NO.187 more than 1 or 1, at the HER-2 gene be selected among the SEQ.NO.188-SEQ.NO.191 more than 1 or 1; At the B2M gene be selected among the SEQ.NO.192-SEQ.NO.199 1 or many and/or among the SEQ.NO.200-SEQ.NO.203 1 or many of being selected from of TFRC gene.

7. detect liquid-phase chip according to claim 5 or 6 described receptor tyrosine kinase inhibitors class targeting medicament curative effect related gene mRNA expression levels, it is characterized in that described spacerarm sequence is 5-10 T.

8. receptor tyrosine kinase inhibitors class targeting medicament curative effect related gene mRNA expression level according to claim 5 detects liquid-phase chip, it is characterized in that, described P5 consists of CCTATGCCTCCCGTGTCTA.

9. a method that detects receptor tyrosine kinase inhibitors class targeting medicament curative effect related gene mRNA expression level is characterized in that, uses each described liquid-phase chip of claim 1-4, mainly may further comprise the steps:

(1) lysed sample discharges total RNA, gets the cracking mixed solution;

(2) cracking mixed solution, coupling there are microballoon, support extension probes, the amplification extension probes of supporting probe are sealed in the hybridization plate, 54 ℃ ± 1 ℃, 600rpm shakes overnight incubation, support the sequence P3 of extension probes to combine with the P1 that supports probe is complementary, support the specific sequence P2 and the target mRNA specific combination of extension probes, thereby target mRNA is attached on the microballoon; The amplification extension probes has biotin labeling, amplifies thereby the P4 of amplification extension probes and target mRNA specific combination realize the cascade of target mRNA signal;

(3) product and the Streptavidin-phycoerythrin of step (two) react;

(4) detect by fluorescence detector.

10. a method that detects receptor tyrosine kinase inhibitors class targeting medicament curative effect related gene mRNA expression level is characterized in that, uses each described liquid-phase chip of claim 5-8, mainly may further comprise the steps:

(1) lysed sample discharges total RNA, gets the cracking mixed solution;

(2) cracking mixed solution, coupling there are microballoon, support extension probes, the amplification extension probes of supporting probe are sealed in the hybridization plate, 54 ℃ ± 1 ℃, 600rpm shakes overnight incubation, support the sequence P3 of extension probes to combine complementary combination with the P1 that supports probe, support the specific sequence P2 and the target mRNA specific combination of extension probes, thereby target mRNA is attached on the microballoon; The P4 and the target mRNA specific combination of amplification extension probes;

(3) add label probe again, label probe has biotin labeling, and 50 ℃ ± 1 ℃, the 600rpm concussion was hatched 1 hour, and label probe combines with the sequence P5 complementary pairing of amplification extension probes, thereby the cascade that realizes target mRNA signal is amplified;

(4) product and the Streptavidin-phycoerythrin of step (three) react;

(5) detect by fluorescence detector.